Angiogenesis refers to a mechanism by which new blood vessels are formed from the pre-existing blood vessels through the growth, differentiation and migration of endothelial cells. It is known that angiogenesis plays an important role in the normal growth process including wound healing and a woman's menstrual cycle (Risau, Nature, 386: 671, 1997) and the abnormally excessive angiogenesis plays a critical role in the growth and metastasis of tumor and the attack of diseases such as age-related macular degeneration (ARMD), diabetic retinopathy, psoriasis, rheumatoid arthritis and chronic inflammation (Carmeliet and Jain, Nature, 407: 249, 2000).
In 1971, Dr. J. Folkman proposed a hypothesis that the growth and metastasis of tumor is angiogenesis-dependent, and thus the therapeutic strategy focusing on the anti-angiogenesis may involve a new therapeutic agent for solid cancer. Thereafter, many researchers have been increasingly interested in excessive research associated with the inhibition of the angiogenesis mechanism (Ferrara and Kerbel, Nature, 435: 967, 2005). The pattern of progression of angiogenesis is determined by the general balance between angiogenesis-inducing factors and angiogenesis-inhibiting factors and takes place through complex and sequential processes of multiple steps. When referring to such a process, first, various angiogenesis-inducing factors including a vascular endothelial growth factor (VEGF) secreted from tissues with tumor or wounds bind to their corresponding receptors in the periphery of pre-existing vascular endothelial cells to activate the vascular endothelial cells, thereby increasing the permeability of the vascular endothelial cells. Moreover, proteinases such as a matrix metalloproteinase (MMP) are secreted to digest the basement membrane and extracellular matrix around the vascular endothelial cells, such that the vascular endothelial cells escape from the pre-existing capillary vessel and migrate/proliferate toward tissues secreting the angiogenesis-inducing factor. The migrating and proliferating vascular endothelial cells form a tubular structure in the blood vessel, and a stable and mature blood vessel is finally formed as a pericyte which is a structural support of the vascular endothelial cells flows into the tubular structure. In this case, it is known that angiopoietin 1 (Ang1) secreted from the vascular endothelial cells plays an important role in the influx of pericyte and the stabilization of blood vessels by binding to its receptor, Tie-2 (Suri et al., Cell, 87:1171, 1996). Meanwhile, angiopoietin 2 (Ang2) which is known to inhibit the interaction of Ang1 and Tie-2 shows similar affinity to Tie-2 compared to Ang1, and thus Ang2 can serve to competitively inhibit a phosphorylation process induced by Ang1 (Maisonpierre et al., Science, 277:55, 1997). However, it was reported that Ang2 functions to induce the phosphorylation of Tie-2 depending on the shape of cells and the experimental method (Kim et al., Oncogene, 19:4549, 2000). Also, it was reported that the blood vessel becomes unstable and the vascular endothelial cells become sensitive to stimuli such as VEGF by inhibiting the interaction between the vascular endothelial cell and pericyte at the early stage of angiogenesis (Klagsbrun and Moses, Chem. Biol., 6:R217, 1999; Veikkola and Alitalo, Semin Cancer Biol., 9:211, 1999; Carmeliet and Jain, Nature, 407:249, 2000). In particular, Ang1 is relatively widely expressed in normal tissues (Maisonpierre et al., Science, 277:55, 1997), but is poorly expressed in tumor tissues (Hayes et al., Br. J. Cancer, 83:1154, 2000). On the other hand, from the fact that Ang2 is over-expressed in cancer tissues having a high angiogenic potential or normal tissues, such as placenta, uterus and ovary, whose blood vessel remodeling actively takes place (Kong et al., Cancer Res., 61:6248, 2001; Ahmad et al., Cancer, 92:1138, 2001), it can be presumed that the onset of angiogenesis in tumor occurs as Ang2 is present at a higher content than Ang1. Therefore, it is supposed that Ang2 serves as an agonist in a Tie-2 signaling mechanism. In sum, since the signaling mechanisms of angiopoietin and Tie-2 are not clearly identified, additional studies are required to identify the signaling mechanisms. However, Ang1 and Ang2 are considered to play important and different roles in angiogenesis.
Angiopoietin includes an N-terminus domain (N-domain) consisting of approximately 50 amino acids, a coiled coil domain (C-domain) consisting of 215 amino acids, and a fibrinogen-like domain (F-domain) consisting of approximately 215 amino acids. Among them, the N- and C-domains are associated with polymerization of angiopoietin, and the F-domain is associated with binding of a receptor Tie-2 (Davis et al., Nat. Strut. Biol., 10:38, 2003). Phosphorylation required to induce signals of Tie-2 is achieved by dimerization of the receptor like other tyrosine kinase receptors. From the fact that angiopoietin should be multimerized for this purpose (Procopio et al., J. Biol. Chem., 274:30196, 1999; Schlessinger, Cell, 103:211, 2000), it is suggested that these ligands may be used as a target for new drug development using the inhibition of angiogenesis. In particular, Davis et al. argued that a minimal module of angiopoietin for the phosphorylation of Tie-2 is a tetramer as measured using a genetic engineering technique and may be used as an antagonist when the module is composed of dimmers (Davis et al., Nat. Strut. Biol., 10:38, 2003). In another study, it was reported that an Ang1 dimeric variant does not effectively perform signaling for Tie-2 (Cho et al., Proc. Natl. Acad. Sci. U.S.A., 101:5547, 2004). That is, although it was not reported that Ang1 serves as an antagonist, the inhibition of signaling for Tie-2 by modifying the oligomeric pattern of this molecule indicates that a strategy for preventing multimerization of angiopoietin may be used to hinder the Tie-2 signaling mechanism. According to the reported binding structure of angiopoietin and Tie-2, it can be seen that a Tie-2 binding site is present in angiopoietin and the binding structure between Ang1 and Tie-2 is similar to that between Ang2 and Tie-2 (Barton W. A. et al., Nat. Struct. Biol., 13:524, 2006). Therefore, the present researchers have tried to effectively inhibit angiogenesis by constructing a dual targeting antibody in which a Tie-2 binding site to angiopoietin, particularly Ang2 in the present invention, is fused to a pre-existing antibody.
Meanwhile, as another target to inhibit angiogenesis, attention has been paid to a VEGF/VEGFR signaling mechanism in the present invention. VEGF that is known to have a significant effect on most steps of an angiogenic process is widely secreted from a hypoxia site of a tumor region. In 1989, VEGF was identified by Dr. N. Ferrara, et al. of Genentech through protein isolation and purification and cDNA cloning (Leung et al., Science, 246:1306, 1989). VEGF called VEGF-A has been known to have four isotypes (VEGF121, VEGF165, VEGF189 and VEGF206). Among them, it was reported that VEGF165 is abundant in all human tissues except for the placenta (Tisher et al., J. Biol. Chem., 266:11947, 1991). It is known that VEGF binds to its receptors VEGFR-1 and VEGFR-2 with a very high affinity but induces mechanisms associated with angiogenesis such as proliferation and migration of vascular endothelial cells by transducing its signals through VEGFR-2. For this reason, VEGF and VEGFR-2 have been mainly targeted to inhibit the angiogenesis mechanisms induced by VEGF, and many articles dealing with these contents were reported (Ellis and Hicklin, Nature Rev. Cancer, 8:579, 2008; Youssoufian et al., Clin. Cancer Res., 13:5544s, 2007). For example, Avastin of Genentech is a humanized antibody targeting VEGF-A (Ferrara et al., Biochem. Biophy. Res. Comm., 333:328, 2005) and has been approved by the U.S. Food and Drug Administration (FDA) for metastatic colon cancer in 2004, non-small cell lung cancer in 2006 and Her-2 negative metastatic breast cancer in 2008, respectively, and come into the market. In recent years, extensive clinical tests on various solid tumors have been conducted to enlarge the indications. In addition, Lucentis commercially available from the same company is an antibody produced by digesting only a Fab fragment from Avastin to promote its permeability when Lucentis is injected into the retina to inhibit excessive angiogenesis under the macula which is a major condition of the senile macular degeneration (Eter et al., Biodrgus, 20:167, 2006) and was approved in 2006 by the USA FDA as a therapeutic agent to treat wet age-related macular degeneration (wet-ARMD). Another therapeutic antibody targeting VEGF is VEGF-trap of Regeneron (Holash et al., PNAS, 99:11393, 2002). This is a water-soluble “decoy receptor” obtained by fusing the second immunoglobulin domain of VEGFR-1 and the third immunoglobulin domain of VEGFR-2 with human Fc and has not been approved by the USA FDA, and its phase III trials for metastatic breast cancer, metastatic lung cancer, metastatic colon cancer and hormone-refractory prostate cancer are now under way.
Angiogenesis-inhibiting antibodies targeting a VEGF receptor VEGFR-2 include IMC-1121B (EP 1916001A2) of Imclone, CDP-791 (PCT/GB02/04619) of UCB, and TTAC-0001 (PCT/KR07/003,077) developed by the present researchers. IMC-1121B is a monoclonal antibody screened from the complete human Fab library, its phase III trials for metastatic breast cancer are now under way, and its phase III trials for gastric cancer is scheduled to be conducted in 2009. CDP-791 of UCB is a humanized antibody, and its phase II trials for non-small cell lung cancer in the form of PEGylated Di-Fab are now under way. Since this antibody does not contain an Fc domain, the antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity cannot be expected. Lastly, TTAC-0001 developed by the present researchers and studied in a pre-clinical stage is a monoclonal antibody screen from the complete human ScFv library. This is only one antibody that shows reactivity to mouse- or rat-derived flk-1 (VEGFR-2 homologue) while targeting VEGFR-2 and is one of the important features differentiating from the IMC-1121B of Imclone (PCT/KR07/003,077). In particular, the cross-species cross reactivity of TTAC-0001 enables the research on an animal disease model, which helps to develop an anti-cancer agent for treatment of specific tumors in the future and to complete the related research more easily.
As such, the research aimed at the VEGF and VEGFR-2 has made great strides for the recent 5 years, and various therapeutic agents have been developed through the market and clinical studies. In addition to developing a therapeutic antibody using the inhibition of angiogenesis, many antibody therapeutic agents using a single target for each disease have been approved by the FDA and come into the market. For example, major antibody therapeutic agents leading the market of monoclonal antibodies over the world include Eribitux (Imclone) that targets an epidermal growth factor receptor (EGFR) and has been marketed as a therapeutic agent to treat metastatic colon cancer, Herceptin (Genentech) that targets Her-2/neu and has been marketed as a therapeutic agent to treat metastatic breast cancer, and Rituxan™ that targets CD-20 and has been marketed as a therapeutic agent to treat non-Hodgkin's lymphoma.
Meanwhile, according to the recent trends of the antibody market, in addition to developing an antibody having the functionality for a single target, extensive research aimed at developing a so-called dual targeting antibody (bispecific antibody) or a multi-targeting antibody (multi-specific antibody), which can have two or more targets at the same time, has been actively conducted (Van Spriel et al., Immunol. Today, 21:391, 2000; Kufer et al., Trend in Biotechnol., 22:238, 2004; Marvin and Zhu, Curr. Opin. Drug Discovery Dev., 9:184, 2006). Among these antibodies belonging to this class, there is no case in which any antibody is approved by the FDA and produced on a commercial scale. However, these antibodies have been steadily studied in laboratory and clinical levels on the basis of the continuous interests and potentials. The antibodies belonging to this class are mainly classified into (1) ScFv-based antibodies, (2) Fab-based antibodies, and (3) IgG-based antibodies, etc.
First, in the case of the ScFv-based multi-targeting antibody, there is a diabody obtained by combining VL and VH of different ScFvs and having a hybrid ScFv in a heterodimeric form (Holliger et al., Proc. Natl. Acad. Sci. U.S.A., 90:6444, 1993). However, such an antibody has a problem of poor stability due to the binding affinity to heterodimers. Also, tendem ScFv obtained by liking different ScFvs to each other (Kipriyanov et al., J. Mol. Biol., 293:41, 1999; Robinson et al., Brit. J. Cancer, 99:1415, 2008), heterodimeric ScFv obtained by expressing Jun and Fos which have a binding potentials to the termini of different ScFvs (De Kruif and Logtenberg, J. Biol. Chem., 271:7630, 1996), a heterodimeric miniantibody obtained by expressing CH1 and CL of Fab from the termini of ScFv, respectively (Muller et al., FEBS lett., 432:45, 1998), and a method for constructing a minibody in a heterodimeric ScFv form (Merchant et al., Nat. Biotechnol., 16:677, 1998) were reported. In this case, the method for constructing the minibody includes substituting some amino acids of a CH3 domain that is a homodimeric domain of Fc to change a heterodimeric structure in a “knob into hole” form and expressing these modified CH3 domains from the termini of ScFv, respectively. In addition, a variety of ScFv-based analogues were reported in the scientific literature (Kipriyanov and Le Gall, Curr. Opin. Drug Discovery Dev., 7:233, 2004), and a triple target ScFv using a triabody and a quadruple target ScFv using a tetrabody were also reported in the scientific literature (Hudson and Kortt, J. Immunol. Methods, 231:177, 1999).
Second, the Fab-based multi-targeting antibody mainly has the form of heterodimeric Fab obtained by combining separate Fab's against specific antigens with each other using a disulfide bond or a mediator (Brennan et al., Science, 229:81, 1985; Kostelny et al., J. Immunol., 148:1547, 1992). Meanwhile, it was reported that a dual targeting antibody having two antigen valencies is produced by expressing ScFvs against different antigens from the termini of a heavy chain or a light chain of specific Fab (Schoonjans et al., J. Immunol., 165:7050, 2000; Lu et al., J. Immunol. Methods, 267:213, 2002), and a dual targeting antibody is produced in a homodimeric form to have four antigen valencies by interposing a hinge region between Fab and ScFv (Coloma and Morrison, Nat. Biotechnol., 15:159, 1997). Also, a dual targeting bibody produced with three valencies for antigens by fusing ScFvs against different antigens to the termini of the light chain and heavy chain of Fab, and a triple target bibody produced with three valencies for antigens by fusing different ScFvs respectively to the termini of the light chain and heavy chain of Fab were also reported in the scientific literature (Schoonjans et al., J. Immunol., 165:7050, 2000). Also, a triple targeting antibody F(ab′)3 produced in a simple shape by chemically conjugating three different Fabs was also reported (Tutt et al., J. Immunol., 147:60, 1991).
Third, in the case of the IgG-based multi-targeting antibody, a hybrid hybridoma producing a dual targeting antibody, so-called quadroma was obtained by Trion Pharma by hybridizing mouse and rat hybridomas. The dual targeting antibody Ertumaxomab (antigen: Her-2/neu, CD3) produced by this company proceeded into phase II trials for metastatic breast cancer (Kiewe and Thiel, Expert Opin. Investig. Drugs, 17:1553, 2008), and Catumaxomab (antigen: EpCAM, CD3) proceeded into phase II trials for gastric cancer and ovarian cancer and phase III trials for malignant ascites (Shen and Zhu, Curr. Opin. Mol. Ther., 10:273, 2008). However, these antibodies cannot exclude a human anti-mouse antibody (HAMA) or human anti-rat antibody (HARA) reaction caused by their repeated administration. Meanwhile, a dual targeting antibody of “Holes and Knob” produced in a heterodimeric form by modifying some amino acids of a CH3 homodimeric domain of Fc for different heavy chains while sharing a light chain domain was also reported (Merchant et al., Nat. Biotechnol., 16:677, 1998). In addition to the dual targeting antibody in a heterodimeric form, it was reported that (ScFv) 4-IgG (antigen: EGFR, IGF-1R) is expressed in a homodimeric form by fusing two different ScFvs to the constant domains of a light chain and a heavy chain of IgG instead of the variable domains thereof (Lu et al., J. Biol. Chem., 279:2856, 2004). However, this antibody has a problem of very low productivity, but the same research group produced a di-diabody with improved productivity to the same target by compensating the problem of (ScFv) 4-IgG, and confirmed its potentials (Lu et al., J. Biol. Chem., 280:19665, 2005). However, such an antibody did not overcome the problem of poor stability of the diabody. Also, Shen et al. of Imclone produced a dual targeting antibody by fusing only a single variable domain for mouse platelet-derived growth factor receptor-α (PDGFR-α) to the N-terminus of a light chain of a chimeric monoclonal antibody IMC-1C11 against human VEGFR-2, and reported its potentials (Shen et al., J. Biol. Chem., 281:10706, 2006; Shen et al., J. Immunol. Methods, 318:65, 2007). Recently, Rossi et al. proposed an antibody having multiple antigen valencies for CD20 by a method so called “dock and lock (DNL)” using a dimerization and docking domain (DDD) of an R subunit of protein kinase A (PKA) and an anchoring domain of the PKA (Rossi et al., Proc. Natl. Acad. Sci. U.S.A., 103:6841, 2006; Rossi et al., Cancer Res., 68:8384, 2008), and the same research group reported a dual targeting antibody on the basis of such a technique (Chang et al., Clin. Cancer Res., 13:5586, 2007). It is known that the antibodies using the DNL method have advantages in that they are easy to apply, can be variously combined since it is produced in a module type, and has excellent in vivo stability, but have problems in that they may be degraded by in vivo proteinase and have immunogenicity-related problems.
There are various dual targeting or multi-targeting antibodies reported in the scientific literature up to the present, and these antibodies have functional merits and demerits depending on the morphological features according to their intended uses. In particular, extensive research aimed at developing therapeutic dual targeting and multi-targeting antibodies to treat cancer has continued to progress. However, it is very important to select antigens targeted by the antibodies obtained through such research such that the antibodies can perform their proper functions.